Early analysis and the possibility of rapidly genotyping the pathogenic agent are among the principal objectives of research in the diagnostic field.
At present, the development of new diagnostic assays should take into consideration the following factors: compatibility to high-throughput screening methods; a higher degree of diagnostic sensitivity in the preliminary phase of the infection; and, particularly in developing countries, the ease of handling of biological samples, for more widespread distribution of their use.
There are currently three types of in vitro diagnostic systems: direct culture of the pathogenic agent from the biological sample, which is the so-called “gold standard” of diagnostic assays; immunological assays based on the detection of products or antigens of the infectious agent; and indirect immunological assays that can detect antibodies produced against the infectious agent during infection.
In the first system, the principal disadvantage is that the biological sample must be considered to be at risk for the transmission of the pathogenic agent, whereas in the latter case, there is no possibility of discriminating between past and current infections.
More recently, molecular diagnostic methods have been developed based on the detection of the nucleic acids of the pathogenic agent in the blood or plasma samples, or in the cell cultures, taken from the patient. These assays are generally much more sensitive than the immunological assays, and for this reason, and for their specificity, they are extremely promising. However, they may require the presence of special equipment and qualified personnel. Furthermore, the biological samples—in the case of plasma, blood, or cell cultures—are difficult to store unaltered, except under controlled temperature conditions.
Recently, molecular diagnostic methods based on transrenal DNA have been described for monitoring the progress of allogeneic transplants, to diagnose the sex of a fetus, and to detect the presence of tumoral markers. In particular, U.S. Pat. No. 6,251,638 describes an analytical method for detecting male fetal DNA in the urine of pregnant women; in U.S. Pat. No. 6,287,820, the system is aimed at the diagnosis of tumors, particularly of adenocarcinomas (of the colon and pancreas); and in U.S. Pat. No. 6,492,144, the Tr-DNA nucleic-acid analysis method is used to monitor the progress of allogeneic transplants, using known methods for the molecular analysis per se. The presence of identifiable transrenal DNA in urine, in fragments of nucleic-acid sequences (consisting of fewer than 1000 DNA base pairs) that cross the renal barrier, was also described in: Al-Yatama et al. (2001), “Detection of Y-chromosome-specific DNA in the plasma and urine of pregnant women using nested polymerase chain reaction” Prenat Diagn, 21:399-402; and in Utting, M., et al. (2002), “Microsatellite analysis of free tumor DNA in urine, serum, and plasma of patients: a minimally invasive method for the detection of bladder cancer”; Clin Cancer Res, 8:35-40.
Molecular detection of TrDNA in urine is performed in the same way as for other types of DNA, e.g., through PCR (polymerase chain reaction), nested PCR, hybridization, SSCP, LCR, SDA, or the so-called “cycling probe reaction”.
The presence of transrenal DNA has been explained through the apoptosis phenomenon. During cell death most of the nuclear DNA is converted into nucleosomes and oligomers (Umansky, S. R., et al. [1982], “In vivo DNA degradation of thymocytes of gamma-irradiated or hydrocortisone-treated rats”; Biochim. Biophys. Acta 655:9-17), which are finally digested by macrophages or neighboring cells. However, a portion of this degraded DNA escapes phagocytic metabolism, and can be found in the bloodstream (Lichtenstein, A. V., et al. [2001], “Circulating nucleic acids and apoptosis”; Ann NY Acad Sci, 945:239-249), and, as confirmed in the above-indicated patents, also in urine.
The application of this system to bacteriological infections is not clear, because it has not been well studied and because prokaryotic DNA package is different from that of eukaryotic DNA. In the prior art, in contrast to the present method, the best-known system consists of isolating prokaryotic DNA from urine sediment that contains bacteria (Frasier, et al. [1992], “DNA probes for detecting Coxiella burnetii strains”; Acta Virol, 36:83-89). What happens to the prokaryotic DNA during infection is also well known. The prokaryotes are ingested by the cells of the immune system, such as macrophages and dendritic cells. The prokaryotes are then dissolved by the phagolysosome vesicles. The prokaryotic DNA is then released by the cell and a portion of this DNA enters the bloodstream in either of two ways: (a) the ingesting cell becomes apoptotic and breaks apart (Navarre, W. V. [2000], “Pathogen-induced apoptosis of macrophages: a common end for different pathogenic strategies”; Cell Microbiol 2:265-273); or (b) the phagolysosome vesicles release the fragments of the prokaryote (including the fragmented DNA) into the bloodstream (Friedlander, A. M. [1978], “DNA release as a direct measure of microbial killing by phagocytes”; Infect Immune 22:148-154).
The conclusion that can be drawn from these studies is that the diagnosis of the prokaryotic infection is made during the initial period of the infection. The same principle applies to parasitic infections, in which even less investigative work has been done on the effect of the infection in relation to the presence of DNA in urine. In the case of malaria, a diagnostic system that can function during the initial periods of the infection is not yet available. However, the need for such a system is recognized. In fact, at present, using current diagnostic systems, it is still difficult to distinguish between the presence of malaria in the initial period and viral hepatitis.